Preparation, structure determination, and in silico and in vitro Elastase inhibitory properties of substituted N-([1,1′-Biphenyl]-2-ylcarbamothioyl)- Aryl/Alkyl benzamide Derivatives

Article abstract of DOI:10.1016/j.molstruc.2021.130993

The preparation of a set of eight closely related biphenyl-thiourea conjugates with aromatic and aliphatic side chains (3a-3h) using a one-pot three-component strategy is reported. All the novel compounds were characterized by spectroscopic techniques (FT

Full text of DOI:10.1016/j.molstruc.2021.130993

Journal of Molecular Structure 1245 (2021) 130993
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Preparation, structure determination, and in silico and in vitro
Elastase inhibitory properties of substituted
ꢀ
Derivatives
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)- Aryl/Alkyl benzamide
Sara Ilyasa, Aamer Saeeda,∗, Qamar Abbasb, Rabail Ujana, Pervaiz Ali Channara,
Izhar Ahmed Shaikhc, Mubashir Hassand, Hussain Razae, Sung-Yum Seoe,
Gustavo A. Echeverríaf, Oscar E. Pirof, Mauricio F. Erbeng,∗
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
b Department of Biology, College of Science, University of Bahrain, Sakhir, 32038 Kingdom of Bahrain
c Department of PCSIR head office ministry of science and technology Islamabad, Pakistan
d Institute of Molecular Biology and Biotechnology/(IMBB), The University of Lahore, 1-KM, Defense Road, Bhubtian Chowk, Lahore, Pakistan
e Department of Biological Sciences, College of Natural Sciences, Kongju National University, 56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of
Korea
f Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata e Instituto de Física La Plata, IFLP (UNLP, CONICET, CCT-La Plata),
C. C. 67, 1900 La Plata, Argentina
g CEQUINOR (UNLP-CONICET, CCT-La Plata), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Boulevard 120 e/ 60
y 64 N 1465 La Plata B1900, Buenos Aires, Argentina
º
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation of a set of eight closely related biphenyl-thiourea conjugates with aromatic and aliphatic
side chains (3a-3h) using a one-pot three-component strategy is reported. All the novel compounds were
characterized by spectroscopic techniques (FTIR, 1H and 13 C NMR) and elemental analysis. Moreover, the
crystal structure of compounds 3f and 3h have been determined by X-ray diffraction. The common molec-
ular skeleton can be closely superposed to each other and the 1-acyl thiourea groups show a nearly pla-
nar conformation favored by an intramolecular N–H•••O=C bond. In-vitro studies were carried out to
test the elastase inhibition activity of the newly synthesized biphenyl-thiourea hybrid derivatives. Among
Received 19 May 2021
Revised 14 June 2021
Accepted 26 June 2021
Available online 14 July 2021
Keywords:
Aryl thioureas
Structural X-ray diffraction
Biological activity
Docking studies
the series, compound 3c (IC50 = 0.26
0.05 μM) exhibited the maximum inhibition against elastase. The
higher activity of aryl substituents over alkyl chains is evidenced, as well as the importance of electron
withdrawing groups, as nitro (3b and 3c) and bromo (3d) to enhance the enzyme inhibitory activity. The
compound 3c inhibits the enzyme in a competitive manner, with dissociation constant Ki = 0.84 μM.
Molecular docking was also carried out within the enzyme active site to study enzyme-inhibitor interac-
tions. Docking results correlate with experimental inhibition studies and show that compound 3c exhibits
the highest binding energy (-7.70 kcal/mol) as compared with other compounds. The results of this study
might help to develop new elastase inhibitors.
Elastase inhibition activity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
forts to explore more such multipurpose thio compounds [2,3].
A wide range of 1-(acyl/aroyl)-3-(substituted) thioureas are ex-
Acyl thiourea compounds are currently receiving considerable
attention because of their wide applications in several fields [1].
The straightforward synthetic route and excellent properties are
the important features which engage many researchers in ef-
tremely versatile starting materials for the synthesis of a vari-
ety of heterocyclic compounds due to the presence of two hy-
drogen atoms bonded to each of the nitrogen atoms [4]. More-
over, an array of pharmacological properties associated with
1-(acyl/aroyl)-3-(substituted)thioureas has made them attractive
templates for future drug design. Thiourea and urea derivatives
show a broad spectrum of biological activities such as antitu-
mor [5], herbicidal [6], antiviral [7], antiparasitic [8], antimi-
∗
Corresponding authors.
E-mail addresses: aamersaeed@yahoo.com (A. Saeed), erben@quimica.unlp.edu.ar
(M.F. Erben).
https://doi.org/10.1016/j.molstruc.2021.130993
0022-2860/© 2021 Elsevier B.V. All rights reserved.

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
ꢀ
crobial [9], insecticidal [10], pesticidal and fungicidal properties
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)-4-methylbenzamide (3a)
[11,12]. Thus, 1-acyl-3-(2-amino-phenyl) thioureas exhibit anti-
´
intestinal nematode activity [13], the thiourea derivatives of 4-
azatricyclo[5.2.2.02,6]undec-8-ene-3,5-dione [14] and pyridazine
[15]; 1-aroyl-3-(substituted-2-benzothiazolyl)thioureas [16] and 1-
aroyl-3-arylthioureas [17] exhibit potent antibacterial activity. N-
Phenyl and N-benzoyl thioureas are antimicrobial agents [18] and
excellent antifungal activity were reported for fluorinated thioureas
[19].
White solid, m.p.: 181-183
°C, Yield= 82 %, Rf= 0.51 (n-Hexane: Ethyl acetate 4:1); IR (KBr,
v/cm−1): 3350 (NH), 3143 (NH), 1650 (C=O), 1579, 1473 (C=C aro-
matic), 1253 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 11.81
(s, 1H, NH), 9.41 (s, 1H, NH), 7.91 (d, 2H, J=7.9 Hz, Ar–H), 7.43
(d, 2H, J=7.8 Hz, Ar–H), 7.32-7.41 (m, 4H, Ar–H), 7.35 (m, 4H, Ar–
H), 7.14 (t, 1H, J=2.1 Hz, Ar–H), 2.36 (s, 3H, CH3); 13C NMR (75
MHz CDCl3); δ (ppm): 185.1 (C=S), 168.4 (C=O), 140.5, 138.6, 137.8,
135.9, 132.0, 131.5, 130.7, 129.1, 128.7, 128.2, 127.9, 127.7, 127.4
(Aromatic-Cs), 21.5 (Ar-CH3).
In addition, biphenyl derivatives are used in the synthesis
of antifungal agents like bifonazole, optical brightening agents,
dyes, and polychlorinated biphenyls (PCBs). In particular, biphenyl
thioureas have been used as organocatalysts for electrochemical re-
ductions [20].
Few structures containing the biphenyl-thiourea scaffold can
be found in the literature, the list includes the N-(biphenyl-4-
carbonyl)-N -(4-chlorophenyl)thiourea [21], its 2-chloro- [22], 2-
´
pyridylmethyl)- [23], and 6-methylpyridin-2-yl [24] analogues. In
these cases, the thiourea scaffold is formed from the corresponding
biphenyl isothiocyanate. Then, as a first objective of this work, we
wanted to know the capability of using the carbonyl isothiocyanate
route for preparing acyl/aryl-carbonyl biphenyl thioureas. A second
aim is to determine how different substituent affects the structural
properties around the central 1-acyl thiourea group [25]. Finally,
the investigation of biological properties is also undertaken in con-
tinuation of our studies on sulfur organic compounds [26,27]. Thus,
considering the aforesaid significance of thiourea motif and the
multifunctional value of biphenyl group, the aim of the current
work was the synthesis of thiourea species containing the biphenyl
group in a single structural entity. Eight close related compounds
were prepared and characterized by FTIR and multinuclear (1H and
13C) NMR spectroscopy. The X-ray single crystal structure of two
compounds were determined. Furthermore, the synthesized com-
pounds were screened against elastase enzyme to examine biolog-
ical activity, rationalized by kinetic measurements and computa-
tional docking studies.
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)-2-nitrobenzamide (3b)
Light yellow solid, m.p.= 190-
191 °C, Yield= 87 %, Rf= 0.47 (n-Hexane: Ethyl acetate 4:1); IR
(KBr, v/cm−1): 3325 (NH), 3165 (NH), 1668 (C=O), 1585, 1462 (C=C
aromatic), 1234 (C=S), 1554, 1338 (N=O); 1H NMR (CDCl3, 300
MHz,); δ (ppm): 11.75(s, 1H, NH), 9.49 (s, 1H, NH), 8.45 (d, 1H,
J=7.5 Hz, Ar–H), 8.29 (m, 2H, Ar–H), 7.57 (dd,1H, J=7.8 Hz, Ar–
H), 7.33-7.40 (m, 4H, Ar–H), 7.37 (m, 4H, Ar–H), 7.15 (t, 1H, J=2.1
Hz, Ar–H); 13C NMR (75 MHz CDCl3); δ (ppm): 180.1 (C=S), 166.4
(C=O), 138.3, 138.1, 136.8, 135.5, 133.0, 131.8, 130.4, 130.2, 129.5,
128.9, 128.4, 128.0, 127.7, 127.6, 127.5, 127.4 (Aromatic-Cs).
2. Experimental
Melting points were recorded using
a digital Gallenkamp
(SANYO) model MPD.BM 3.5 apparatus and are uncorrected. 1H
and 13C NMR spectra were determined in CDCl3 at 300 MHz and
75.4 MHz, respectively, using a Bruker spectrophotometer. FTIR
spectra were recorded on an FTS 3000 MX spectrophotometer. Ele-
mental analyses were conducted using a LECO-183 CHNS analyzer.
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)-3,5-dinitrobenzamide (3c)
2.1. General Procedure for the synthesis
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)-4-alkyl/arylbenzamides (3a-3h)
In freshly prepared solution of potassium thiocyanate (1.65
mmol) in dry acetone (15 ml) solution of substituted acid chlo-
ride (1.1 mmol) (1) was added dropwise in a round bottom flask.
Then refluxed the reaction mixture for 2h under nitrogen atmo-
sphere. Milky appearance indicated the acyl thiocyanate formation.
The reaction mixture could cool at room temperature and a solu-
tion of 2-aminobiphenyl (1.1 mmol) in acetone was added drop-
wise in the reaction mixture and refluxed it again for 3h. On the
reaction completion, monitored by TLC, the reaction mixture was
cooled to room temperature and then poured into crushed ice. The
precipitated thioureas were filtered dried and recrystallized from
acetone.
Light brown solid, m.p.=
200-201 °C, Yield= 81 %, Rf= 0.42 (n-Hexane: Ethyl acetate 4:1);
IR (KBr, v/cm−1): 3321 (NH), 3155 (NH), 1659 (C=O), 1575, 1460
(C=C aromatic), 1224 (C=S), 1559, 1340 (N=O); 1H NMR (CDCl3,
300 MHz,); δ (ppm): 12.11(s, 1H, NH), 9.65 (s, 1H, NH), 8.91 (d,
2H, J= 2.1 Hz, Ar–H), 8.86 (s, 1H, Ar–H), 7.30-7.40 (m, 4H, Ar–H),
7.35 (m, 4H, Ar–H), 7.14 (t, 1H, J= 2.1 Hz, Ar–H); 13C NMR (75
MHz CDCl3); δ (ppm): 180.1 (C=S), 166.4 (C=O), 138.3, 138.1, 136.8,
135.5, 133.0, 131.8, 130.4, 130.2, 129.5, 128.9, 128.4, 128.0, 127.7,
127.6, 127.5, 127.4 (Aromatic-Cs).
2

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
ꢀ
v/cm−1): 3325 (NH), 3185 (NH), 1659 (C=O), 1572, 1463 (C=C aro-
matic), 1230 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 12.16 (s,
1H, NH), 10.39 (s, 1H, NH), 7.29-7.44 (m, 4H, Ar–H), 7.35 (m, 4H,
Ar–H), 7.61 (d, 1H, J= 7.1 Hz, Ar–H), 2.33 (t, 2H, J= 2.3), 1.57 (q,
2H), 1.29 (m, 6H), 0.91 (t, 3H); 13C NMR (75 MHz CDCl3) δ (ppm)
180.8 (C=S), 174.8 (C=O), 138.5, 138.0, 135.6, 130.1, 128.9, 128.3,
128.2, 127.6, 127.5, 127.5, 127.3 (Aromatic-Cs), 35.8, 26.7, 21.8, 13.1
(n-heptyl, C).
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)-2-bromobenzamide (3d)
Greyish black solid, m.p.= 188-
189 °C, Yield= 82 %, Rf = 0.40 (n-Hexane: Ethyl acetate 4:1); IR
(KBr, v/cm−1): 3315 (NH), 3105 (NH), 1651 (C=O), 1577, 1465 (C=C
aromatic), 1230 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 11.95
(s, 1H, NH), 9.43 (s, 1H, NH), 7.91 (d, 2H, J= 7.9 Hz, Ar–H), 7.76
(d, 2H, J= 7.5 Hz, Ar–H), 7.57-7.59 (m, 2H, Ar–H), 7.27-7.39 (m, 4H,
Ar–H), 7.33 (m, 4H, Ar–H), 7.12 (t, 1H, J= 2.1 Hz, Ar–H); 13C NMR
(75 MHz CDCl3); δ (ppm): 185.1 (C=S), 168.4 (C=O), 138.3, 138.1,
136.8, 135.5, 133.0, 131.8, 130.4, 130.2, 129.5, 128.7, 128.2, 128.0,
127.6, 127.4, 127.1, 126.8 (Aromatic-Cs).
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)octanamide (3h)
Colorless crystals, m.p.= 123-125
°C, Yield= 82 %, Rf= 0.54 (n-Hexane: Ethyl acetate 4:1); IR (KBr)
v/cm−1): 3331 (NH), 3181 (NH), 1659 (C=O), 1580, 1470 (C=C aro-
matic), 1227 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 12.19
(s, 1H, NH), 10.45 (s, 1H, NH), 7.29-7.41 (m, 4H, Ar–H), 7.33 (m,
4H, Ar–H), 7.60 (d, 1H, J= 7.1 Hz, Ar–H), 2.35 (t, 2H, J= 2.3), 1.18
(q, 2H), 1.62 (q, 2H), 1.31 (m, 6H) 0.89 (t, 3H, CH3); 13C NMR (75
MHz CDCl3) δ (ppm) 180.8 (C=S), 174.8 (C=O), 138.5, 138.0, 135.6,
130.1, 128.9, 128.3, 128.2, 127.6, 127.5, 127.5, 127.3 (Aromatic-Cs),
35.8, 26.7, 21.8, 13.1 (n-octyl, C).
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)butyramide (3e)
Grey solid, m.p.= 95-97 °C, Yield=
85 %, Rf= 0.51 (n-Hexane: Ethyl acetate 4:1); IR (KBr, v/cm−1):
3325 (NH), 3179 (NH), 1681 (C=O), 1583, 1460 (C=C aromatic),
1229 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 12.12 (s, 1H,
NH), 10.37 (s, 1H, NH), 7.35-7.42 (m, 4H, Ar–H), 7.39 (m, 4H, Ar–
H), 7.79 (d, 1H, J= 7.1 Hz, Ar–H), 2.36 (t, 2H, J= 2.3), 2.26 (q, 2H),
0.97 (t, 3H); 13C NMR (75 MHz CDCl3) δ (ppm) 180.9 (C=S), 174.5
(C=O), 138.7, 138.2, 135.8, 135.5, 130.2, 128.5, 128.3, 128.2, 127.6,
127.5, 127.4 (Aromatic-Cs), 37.2, 24.4, 13.2 (n-butyl, C).
2.2- In vitro Methodology
2.2.1-Elastase inhibition assay
The elastase (from porcine pancreas) activity was determined
following previously revealed technique by [28-29] with few ad-
justments. To play out the restraint of elastase movement, the
measure of delivered p-nitroaniline, which was hydrolyzed from
the substrate (N-succinyl-Ala-Ala-Ala-p-nitroanilide) by elastase,
was dictated by estimating the absorbance at 410 nm. In detail,
0.8 mM arrangement of N-succinyl-Ala-Ala-Ala-p-nitroanilide was
set up in a 0.2 M Tris-HCl cushion (pH 8.0) and this support (130
μL) was added to the test (10 μL) in a 96 well microplate. The
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)pentanamide (3f)
microplate was pre-hatched for 10 min at 25 C before an elas-
º
tase (0.0375 Unit/mL) stock arrangement (10 μL) was added. Af-
ter catalyst expansion, the microplate was kept at 25 C for 30
º
min, and the absorbance was estimated at 410 nm utilizing mi-
croplate reader (SpectraMax ABS, USA). IC50 esteems were deter-
mined by nonlinear relapse utilizing GraphPad Prism 5.0 (Graph-
Pad, San Diego, CA USA). All tests were done in three-fold. The
elastase hindrance exercises were determined by the accompany-
ing recipe:
Colorless crystals, m.p.= 103-104
°C, Yield= 80 %, Rf= 0.53 (n-Hexane: Ethyl acetate 4:1); IR (KBr,
v/cm−1): 3321 (NH), 3187 (NH), 1685 (C=O), 1581, 1454 (C=C aro-
matic), 1224 (C=S); 1H NMR (CDCl3, 300 MHz,); δ (ppm): 12.14 (s,
1H, NH), 10.38 (s, 1H, NH), 7.36-7.45 (m, 4H, Ar–H), 7.40 (m, 4H,
Ar–H), 7.81 (d, 1H, J= 7.1 Hz, Ar–H), 2.46 (t, 2H, J= 2.3), 1.57 (q,
2H), 1.33 (q, 2H), 0.90 (t, 3H); 13C NMR (75 MHz CDCl3) δ (ppm)
180.8 (C=S), 174.8 (C=O), 138.5, 138.0, 135.6, 130.1, 128.9, 128.3,
128.2, 127.6, 127.5, 127.5, 127.3 (Aromatic-Cs), 35.8, 26.7, 21.8, 13.1
(n-pentyl, C).
ꢀ
ꢁ
Elastase inhibition activity
%
( )
=
ODcontrol − ODsample × 100 /ODcontrol
Where ODcontrol and ODsample signifies the optical densities in
the lack and existence of sample, respectively. Oleanolic acid was
used as the standard inhibitor for elastase.
ꢀ
N-([1,1 -Biphenyl]-2-ylcarbamothioyl)heptanamide (3g)
2.2.2-Protocol for Kinetics
Kinetic analysis was carried out to determine the mode of inhi-
bition by following our optimized method [30]. The compound 3c
was selected on the basis of its high IC50 value. Kinetics were car-
ried out by varying the concentration of N-succinyl-Ala-Ala-Ala-p-
nitroanilide from 2, 1, 0.5, 0.25, 0.125 and 0.0625 mM in the pres-
ence of different concentrations of compound 3c (0.00, 0.25 and
0.51 μM) following the same procedure for the kinetic study as
described in elastase inhibition assay protocol. Maximal initial ve-
locities were determined from initial linear portion of absorbances
Colorless crystals, m.p.= 115-117
°C, Yield= 82 %, Rf= 0.54 (n-Hexane: Ethyl acetate 4:1); IR (KBr,
3

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
up to 10 minutes after addition of enzyme at per minute inter-
val. The inhibition type on the enzyme was assayed by Lineweaver-
Burk plot of inverse of velocities (1/V) versus inverse of substrate
concentration 1/[S] mM−1. The EI dissociation constant Ki was de-
termined by secondary plot of 1/V versus inhibitor concentration.
The results were processed by using SoftMaxPro.
keenly observed to obtain the best docking results. The generated
docked complexes were evaluated based on lowest binding energy
(kcal/mol) values and binding interaction pattern between ligands
and receptor. The graphical depictions of all the docked complexes
were accomplished by UCSF Chimera 1.10.1 and Discovery Studio
(2.1.0), respectively.
2.3-X-ray diffraction data
3- Results and Discussion
The measurements were performed on an Oxford Xcalibur, Eos,
3.1. Synthesis and Characterization
Gemini CCD diffractometer with graphite-monochromated MoKα
˚
(λ= 0.71073 A) radiation. X-ray diffraction intensities were col-
In Scheme 1 the synthetic pathway for aliphatic and aro-
ꢀ
lected (ω scans with ϑ and κ-offsets), integrated and scaled with
CrysAlisPro [31] suite of programs. The unit-cell parameters were
obtained by least-squares refinement (based on the angular set-
tings for all collected reflections with intensities larger than seven
times the standard deviation of measurement errors) using CrysAl-
isPro. Data were corrected empirically for absorption employing
the multi-scan method implemented in CrysAlisPro. The structures
were solved by intrinsic phasing with SHELXT [32] and the non-
H molecular model refined with SHELXL [33]. All hydrogen atoms
of 3f but the methyl ones were located in a difference Fourier
map and refined at their found positions with isotropic displace-
ment parameters. The methyl H-atoms were positioned geometri-
cally and refined with the riding model, treating the –CH3 moi-
eties as rigid groups allowed to rotate around the C–CH3 bonds
such as to maximize the sum of the residual electron density at
the hydrogen calculated positions. Because libration disorder of the
pendant alkane arm, C-C bond distances of 3h were restrained
to be equal during the refinement. All H-atoms were located at
their expected positions and refined with the riding model. Crys-
tal data and structure refinement results are summarized in Tables
S1-S7 (Supporting Information). Crystallographic structural data
have been deposited at the Cambridge Crystallographic Data Cen-
tre (CCDC) with reference numbers CCDC 2058591 (3f) and CCDC
2058592 (3h).
matic derivatives N-([1,1 -biphenyl]-2-ylcarbamothioyl)-substituted
ꢀ
benzamide and N-([1,1 -biphenyl]-2-ylcarbamothioyl)-alkylamide
derivatives is described. Reaction of freshly prepared acyl/aryl 1-
carbonyl isothiocyanates with 2-aminobiphenyl furnished the tar-
get compounds in good yields (higher than 80 %) and high purity.
The synthetized thiourea derivatives are characterized by their
physical parameters, FTIR and NMR spectroscopic techniques. In
the infrared spectra, the characteristic peaks for N-H stretching ap-
pear as broad absorptions in the range of 3355-3185 cm−1. This
feature is in agreement with previous reports on vibrational prop-
erties of 1-acyl-3-monosubtituted thioureas, with the N-H stretch-
ing modes of the thioamide group being shifted to lower frequen-
cies due to the formation of intramolecular N-H•••O=C hydrogen
bond [25]. This interaction also affects the C=O stretching mode,
which is observed as intense and well-defined band in the range
1685-1650 cm−1 [26]. The biphenyl group is characterized by the
presence of aromatic C=C stretching frequencies are observed at
1585-1473 cm−1 and the C=S group absorbs at 1253-1227 cm−1
.
In 1H NMR spectra, the two characteristic N-H protons signals
appeared in the range of δ= 12.16-11.75 and δ= 10.45-9.41 ppm.
The N-H proton connected to the electrophilic functional groups
(carbonyl and thiocarbonyl) is more deshielded as compare to the
N-H proton is connected to the thiocarbonyl moiety [27]. The low-
field resonance observed for the thioamide proton is a signature
for the occurrence of intramolecular N-H•••O=C hydrogen inter-
action, as discussed below. The aromatic region protons were ob-
served from δ= 7.86-7.35 ppm and the aliphatic region protons sig-
nals appeared in the range of δ= 2.29-0.91 ppm, as expected.
In 13C NMR, two differentiating signals of thiocarbonyl and car-
bonyl carbons appeared at δ= 185.1-179.6 and δ= 174.8-165.4 ppm,
respectively. The difference in shift values between C=O and C=S
groups is due to the presence of the NH groups that exert electron
withdrawing effect. The aromatic carbon nucleus of the byphenyl
group resonates in the range ca. 138-126 ppm and the alkyl chains
are observed at higher fields, as expected.
2.4- Computational Methodology
2.4.1-Retrieval of porcine pancreatic elastase structure.
The crystal structure of porcine pancreatic elastase was ac-
cessed from Protein Data Bank (PDB) (www.rcsb.org) with PDBID
7EST and the protein structure was minimized by using UCSF
Chimera 1.6rc [34-35]. The stereo-chemical properties of elastase
and Ramachandran plot and values were also accessed from PDB.
The hydrophobicity graph of target protein was generated by Dis-
covery Studio 4.1 Client [36].
2.4.2- Designing of synthesized compounds and molecular docking
The structures of compounds (3a-h) were sketched in
ACD/ChemSketch and the UCSF Chimera 1.10.1 tool was employed
to energy minimization of each ligand separately having default
parameters such as steepest descent steps 100 with step size 0.02
3.2- Structural results
ORTEP [38] plots of the solid state 3f and 3h molecules are
shown in Figure 1 and corresponding selected bond distances and
angles are reported in Table 1. The 3f-containing solid incorpo-
rated an acetone solvent molecule sited on a crystallographic two-
fold axis and therefore it should be formulated as 3f.½CH3(CO)CH3.
As expected, both compounds show closely related molecular
structures, only differing in the length of the alkyl pendant arm.
In fact, the rms deviation between homologous non-H atoms in
the best least-squares structural fitting of the common skeletal
framework (calculated by the Kabsh procedure [39]) is less than
˚
˚
(A), conjugate gradient steps 100 with step size 0.02 (A) and up-
date interval was fixed at 10. Finally, Gasteiger charges were added
using Dock Prep in ligand structure to obtain the good structure
conformation. Molecular docking experiment of compounds (3a-h)
against the porcine pancreatic elastase were done by using vir-
tual screening tool PyRx with VINA Wizard approach [37]. The
grid box parameters values in VINA search space (X= 4.6898, Y=
57.6015 and Z= -5.7255) were adjusted with default exhaustive-
ness value = 8 to maximize the binding conformational analysis.
We have adjusted sufficient grid box size on biding pocket residues
to allow the ligand to move freely in the search space. All the
synthesized ligands were docked separately against target protein.
In all docked complexes, the ligands conformational poses were
˚
0.15 A. An intramolecular N1H•••O bond [d(N•••O) = 2.613(3)
˚
˚
A and ࢬ(N-H•••O)=140(2) for 3f and d(N•••O) = 2.663(3) A
º
and ࢬ(N-H•••O) = 136 for 3h] promotes a nearly planar 1-acyl
º
thiourea group, as usual for mono-substituted 1-acyl thioureas
[40,41]. Within this group of the better refined 3f compound, ob-
˚
served d(Cph-N) = 1.418(3) A, SC-N bond distances of 1.325(3) and
4

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
Scheme 1. Synthetic route and structures of the thiourea derivatives (3a-h).
Table 1
Selected
ꢀ
bond
lengths
[Ả]
and
angles
[°]
in
N-([1,1 -Biphenyl]-2-
ꢀ
ylcarbamothioyl)pentanamide (3f) in 3f.½CH3(CO)CH3 and N-([1,1 -Biphenyl]-2-
ylcarbamothioyl)octanamide (3h).a
0Parameter
3f
3h
Parameter
3f
3h
C14=O1
C14-N2
C13-N2
C13=S1
C13-N1
N1-C1
1.223(3)
1.378(3)
1.381(3)
1.672(2)
1.325(3)
1.418(3)
1.490(4)
1.229(3)
1.359(3)
1.402(3)
1.659(2)
1.324(3)
1.428(3)
1.498(4)
O1=C14-N2
O1=C14-C15
C14-N2-C13
N2-C13=S1
N2-C13-N1
S1=C13-N1
C13-N1-C1
122.0(2)
121.9(2)
128.1(2)
118.0(2)
115.9(2)
124.5(2)
126.6(2)
122.2(2)
122.2(2)
129.4(2)
118.1(2)
115.5(2)
126.4(2)
125.3(2)
C14-C15
5

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
Fig. 1. Drawings of solid state 3f and 3h molecules showing the labeling of the non-H atoms and their displacement ellipsoids at the 30% probability level. The intra-
molecular H-bonds are indicated by dashed lines.
Fig. 2. Lineweaver–Burk plots for inhibition of elastase in the presence of compound 3c (A). Concentrations of 3c used were 0.00, 0.25 and 0.51 μM. Substrate N-succinyl-
Ala-Ala-Ala-p-nitroanilide concentrations were 2, 1, 0.5, 0.25, 0.125 and 0.0625 mM. (B) The insets represent the plot of the slope.
Table 2
˚
˚
1.381(3) A, and d(OC-N) = 1.378(3) A confirm the single-bond char-
IC50 values (in μM) for the elastase (elastase from porcine pancreas) in-
hibitory activity of derivatives (3a-3h).
acter of C-N links. Double C=S and C=O bond lengths are 1.672(2)
˚
and 1.223(3) A, respectively.
Compound
IC50 SEM (μM)
Compound
IC50 SEM (μM)
The crystals are further stabilized by intermolecular H-bonds.
In fact, 3h is arranged as centrosymmetric dimers, linked
3a
0.915
0.284
0.255
0.518
13.451
0.614
0.077
0.049
0.471
0.014
3e
3f
1.328
0.979
0.997
0.942
0.993
0.655
0.847
0.881
3b
˚
through N2–H•••
S
bonds [d(N•••S’)
=
3.447(2)
A
and ࢬ(N–
3c
3g
3h
H•••S’) = 171(2) ] given rise to a R crystal motif [42]. Neighboring
º
3d
molecules in the 3f lattice are linked to each other through N2–
Oleanolic Acid
˚
H
•••O bonds [d(N•••O’) = 2.874(3) A and ࢬ(N–H•••O’) = 156.5 ],
º
SEM = Standard error of the mean; values are expressed in mean
SEM.
giving rise to a polymeric chain that extends along the crystal c-
axis. These H-bonding structures are further detailed in the sup-
plementary Information (Tables S6 a and b, for 3f and 3h, respec-
tively).
fective inhibitors those with electron donating methyl group (3a).
In particular, the influence of nitro groups in the activity of com-
pounds 3b and 3c will be further determined by using docking
analysis.
a
For atom numbering see Figure 1.
3.3- In-vitro Inhibitory Activity
3.4-Kinetic Studies
All of the synthesized compounds were tested in vitro for their
elastase inhibitory activity. Five different concentrations were used
for calculation of IC50 (Table 2), including the standard oleanolic
acid. It is evident from the IC50 values that in general compounds
bearing aryl groups (3a-3d) have superior activity than those hav-
ing alkyl substitution (3e-3h). Thus, the IC50 values of 0.284
The kinetic study was performed to understand the inhibitory
mode of compound 3c against elastase. Based upon results of IC50
data the most potent compound 3c was selected for determina-
tion of inhibition type and inhibition constant. The kinetic results
of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate
N-succinyl-Ala-Ala-Ala-p-nitroanilide 1/[S] in the presence of dif-
ferent inhibitor concentrations gave a series of straight lines, the
result for compound 3c showed that Vmax remains the same with-
out significantly effecting the slopes. Km increases with increasing
concentration while Vmax remains the same only with minor dif-
0.077 and 0.255
0.049 μM are determined for most active 3b
and 3c compounds, respectively, whereas the IC50 for the alky-
lated thioureas 3a-3d are in the range 0.9-1.3 μM. The analysis of
the substituent effect shows that presence of electron withdrawing
substituents such as nitro (3b and 3c) and bromo (3d) are more ef-
6

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
Fig. 3. A, B. Binding pocket of elastase with embedded ligand 3c.
Fig. 4. Molecular docking interaction of 3c against receptor molecule. A) The general overview of docking depiction. B) The closer view of binding pocket interaction with
best conformation position of ligand against target protein. The interacted amino acids are highlighted in green color and red dotted lines with distance mentioned in
˚
angstrom (A) are justified for H-bond distances.
Table 3
ference. This behavior indicates that 3c compound inhibits the en-
zymes in a competitive manner (Figure 2. A), second plot (Figure 2.
B) of slope against concentration of 3c showed EI dissociation con-
stant. The Ki calculated from inhibitor concentration of 3c versus
the slope was found to be 0.84 μM.
The docking energy values.
Docking Complexes
Binding Affinity (kcal/mol)
3a
3b
3c
3d
3e
3f
-6.7
-7.6
-7.7
-6.7
-5.9
-6.3
-6.6
-6.6
3.5- Molecular docking analysis
Porcine pancreatic elastase consists of 240 amino acids having
calcium atom inserted in the protein structure. The Ramachandran
graph shows 88.70 % of residues were present in favored region
whereas 98.70% residues remain in allowed region. The Ramachan-
dran plot of porcine pancreatic elastase is revealed in supplemen-
tary data Fig S1.
3g
3h
To predict the top suited conformational position of synthesized
compounds (3a-h) within the active region of elastase enzyme,
docking studies were carried. The results indicated that all com-
pounds possess energy values in the range -5.9 to -7.6 Kcal/mol
(Table 3). The docking energy values were calculated by employing
equation 1:
7

S. Ilyas, A. Saeed, Q. Abbas et al.
Journal of Molecular Structure 1245 (2021) 130993
ꢀGbinding = ꢀGgauss + ꢀGrepulsion + ꢀGhbond
+ꢀGhydrophobic + ꢀGtors
Declaration of Competing Interest
(eq. 1)
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Here, ࢞Ggauss: attractive term for dispersion of two gaussian
functions, ࢞Grepulsion: square of the distance if closer than a
threshold value, ࢞Ghbond: ramp function - also used for inter-
actions with metal ions, ࢞Ghydrophobic: ramp function, ࢞Gtors:
proportional to the number of rotatable bonds.
CRediT authorship contribution statement
Sara Ilyas: Data curation, Formal analysis, Investigation,
Methodology, Software, Validation, Visualization. Aamer Saeed:
Conceptualization, Data curation, Formal analysis, Funding ac-
quisition, Investigation, Methodology, Project administration, Re-
sources, Software, Supervision, Validation. Qamar Abbas: Data cu-
ration, Formal analysis, Investigation, Methodology, Software, Val-
idation, Visualization. Rabail Ujan: Data curation, Formal analy-
sis, Investigation, Methodology, Software, Validation, Visualization.
Pervaiz Ali Channar: Data curation, Formal analysis, Investigation,
Methodology, Software, Validation, Visualization. Izhar Ahmed
Shaikh: Data curation, Formal analysis, Investigation, Methodology,
Software, Validation, Visualization. Mubashir Hassan: Data cura-
tion, Formal analysis, Investigation, Methodology, Software, Vali-
dation, Visualization. Hussain Raza: Data curation, Formal anal-
ysis, Investigation, Methodology, Software, Validation, Visualiza-
tion. Sung-Yum Seo: Data curation, Formal analysis, Investiga-
tion, Methodology, Software, Validation, Visualization. Gustavo A.
Echeverría: Data curation, Formal analysis, Investigation, Method-
ology, Resources, Software, Validation, Visualization. Oscar E. Piro:
Data curation, Formal analysis, Investigation, Methodology, Re-
sources, Software, Validation, Visualization. Mauricio F. Erben:
Conceptualization, Data curation, Formal analysis, Funding acquisi-
tion, Investigation, Methodology, Project administration, Resources,
Software, Supervision, Validation.
In docking results compounds having more than 2.5 kcal/mol
energy are thought as potent for inhibition of target enzyme. The
standard error for Autodock is reported as 2.5 kcal/mol (http://
autodock.scripps.edu/). Present docking results vindicated that the
energy value difference among all docking complexes were smaller
than standard error. Therefore, based on both in vitro and in silico
docking energy results, compound 3c was rated as the best lig-
and with good inhibitory potential compared to rest of the deriva-
tives here studied and related compounds already reported [43].
Given that the basic scaffold of all the synthesized compounds was
identical consequently, most of ligands possess good energy values
without any major difference.
3.6- Binding pocket and ligand conformation
The binding pocket analysis revealed that compound 3c binds
in excellent conformational position and best fitted in the bind-
ing pocket of elastase. The amino group on the benzene ring in 3c
showed their binding pattern in the deep region of binding cavity,
whereas rest of benzene rings were confined in the central part of
binding pocket (Figure 3 A, B).
The docked complexes were analyzed based on H-bonding and
hydrophobic contacts. The best in vitro result compound (3c) was
selected to check best conformational position inside active region
of target protein. As shown in Figure 4, the ligand retains nearly
the same conformation in the protein pocket. The 3c form three H-
bonds with different residues of the protein: sulfur forms H-bonds
Acknowledgments
Argentinean authors thank the CONICET (Grant PIP 0651), the
ANPCyT (PICT-2019-2578) and UNLP (Grants 11/X709, 11/X857, and
11/X794) for financial support. GAE, MFE, and OEP are Research
Fellows of CONICET.
˚
with Ser195 with bond length of 2.65 A and the oxygen atoms
of nitro groups form two H-bonds with Ser195 and Val216 having
˚
bonds distance 2.49 and 2.03 A, respectively in line with reported
data [44]. The rest of docking complexes are given in supplemen-
tary data (Fig S2-S8).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130993.
4-Conclusions
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